Rare earth spatial/spectral barcodes for multiplexed biochemical testing

ABSTRACT

Hydrogel microparticles spatially and spectrally encoded using upconverting phosphor nanoparticles are described for use in biochemical testing. In each microparticle, upconversion nanocrystals having spectrally distinguishable emission spectra are disposed in different partions of an encoding region of the microparticle.

RELATED APPLICATIONS

The present application claims benefit of, and priority to U.S. patentapplication Ser. No. 14/214,594, filed Mar. 14, 2014, which claims thebenefit of U.S. Provisional Patent Application No. 61/801,351, filedMar. 15, 2013, and U.S. Provisional Patent Application No. 61/800,995,filed Mar. 15, 2013, all of the above applications being hereinincorporated by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Contract no.FA8721-05-C-0002 awarded by the U.S. Air Force and under Grant Nos.DMR-1006147 and CMMI-1120724 awarded by the National Science Foundation.The government has certain rights in the invention.

BACKGROUND

There are many different approaches currently being employed forperforming multiplexed assays (e.g., two dimensional surface adsorbedarrays, fluorophore-bead systems, spatially-labeled microparticlesystems). The multiplexing capabilities of these techniques may beinsufficient to simultaneously probe some complex, heterogeneousbiological systems.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 schematically depicts an exemplary microparticle, in accordancewith an embodiment.

FIG. 2 is a graph of an emission spectrum of exemplary upconversionnanocrystals (UCNs) labeled “UCN1”, in accordance with an embodiment.

FIG. 3 is a graph of an emission spectrum of exemplary UCNs labeled“UCN2”, in accordance with an embodiment.

FIG. 4 is a graph of an emission spectrum of exemplary UCNs labeled“UCN3”, in accordance with an embodiment.

FIG. 5 is a graph of an emission spectrum of exemplary UCNs labeled“UCN4”, in accordance with an embodiment.

FIG. 6 is a graph of an emission spectrum of exemplary UCNs labeled“UCN5”, in accordance with an embodiment.

FIG. 7 is a graph of an emission spectrum of exemplary UCNs labeled“UCN6”, in accordance with an embodiment.

FIG. 8 is a graph of an emission spectrum of exemplary UCNs labeled“UCN7”, in accordance with an embodiment.

FIG. 9 is a graph of an emission spectrum of exemplary UCNs labeled“UCN8”, in accordance with an embodiment.

FIG. 10 is a graph of an emission spectrum of exemplary UCNs labeled“UCN9”, in accordance with an embodiment.

FIG. 11 is a graph of an emission spectrum of exemplary UCNs labeled“UCN10”, in accordance with an embodiment.

FIG. 12 is a graph of spectral responsivity of RGB channels of a CCDimage sensor with UCN emission bands overlaid, in accordance with anembodiment.

FIG. 13 is a graph of the emission spectrum of UCN6 overlaying thespectral responsivity of RGB channels of a CCD image sensor, inaccordance with an embodiment.

FIG. 14 is a graph showing unique upconversion emission spectra producedby varying dopant concentrations, in accordance with an embodiment.

FIG. 15 is an image of different types of UCNs under NIR illumination,in accordance with an embodiment.

FIG. 16 is a transmission electron micrograph of different types ofUCNs, in accordance with an embodiment.

FIG. 17 includes graphs of emission spectra for different batches ofUCNs, in accordance with an embodiment.

FIG. 18 includes luminescence images of UCNs in liquid with and withoutan applied external magnetic field, in accordance with an embodiment.

FIG. 19 is a graph of magnetization versus applied magnetic field forUCN4, in accordance with an embodiment.

FIG. 20 is a block diagram schematically representing a method offorming a microparticle, in accordance with an embodiment.

FIG. 21 schematically depicts a stop flow lithographic method of forminga contiguous microparticle, in accordance with an embodiment.

FIG. 22 is a luminescence image of microparticles having differentnumbers of encoded regions, in accordance with some embodiments.

FIG. 23 includes graphs of integrated intensity values formicroparticles, each including a different type of UCNs, in accordancewith some embodiments.

FIG. 24 is a scatter plot of integrated intensity data formicroparticles including different types of nanocrystals, in accordancewith some embodiments.

FIG. 25 is a plot of mean measured integrated intensity data andexpected integrated intensity date for the red channel versus the greenchannel showing five-sigma confidence contours, in accordance with anembodiment.

FIG. 26 shows integrated intensity data for different batches ofmicroparticles, in accordance with some embodiments.

FIG. 27 is a graph of emission spectra of UCN4 after each step insurface chemical modification of the UCNs, in accordance with someembodiments.

FIG. 28 shows microparticle emission intensity as a function of timeduring intense sustained NIR irradiation, in accordance with someembodiments.

FIG. 29 shows graphs of intensity versus microparticle age formicroparticles with carboxyl-terminated UCN and for microparticles withacrylated UCN, in accordance with some embodiments.

FIG. 30 is a graph of integrated intensity for different color channelsfor microparticles, in accordance with an embodiment.

FIG. 31 schematically depicts detection of a nucleic acid of interest bya molecular recognition element in the probe region, in accordance withsome embodiments.

FIG. 32 includes images of microparticles having two different codesunder NIR illumination, in accordance with an embodiment.

FIG. 33 includes a bright field image and a fluorescence image of anon-encoded standard microparticle, in accordance with an embodiment.

FIG. 34 is a graph of fluorescence intensity for encoded microparticlesand for standard (non-encoded) microparticles, in accordance with anembodiment.

FIG. 35 is a table with images of encoded microparticles used formultiplexed assays, in accordance with an embodiment.

FIG. 36 is a graph of integrated intensity data in red versus greenchannels for portions or stripes of encoded PEG microparticles.

FIG. 37 is a graph of integrated intensity data in red versus greenchannels for portions or stripes of encoded PUA microparticles andencoded PEG microparticles.

FIG. 38 is an image of microparticles with a PUA encoded region and aPEG-DA probe region under NIR illumination, in accordance with anembodiment.

FIG. 39 is a block diagram of a method for performing biochemical orchemical assay, in accordance with an embodiment.

FIG. 40 includes images of a process of reading out spectral codes froma luminescence image of a microparticle, in accordance with someembodiments.

FIG. 41 includes images used to distinguish two different codes ofmicroparticles, in accordance with an embodiment.

FIG. 42 schematically depicts a flow lithography and decoding system forparticle synthesis, in accordance with some embodiments.

FIG. 43 is an image of the system for particle synthesis of FIG. 42.

Additional features, functions and benefits of the disclosed methods,systems and media will be apparent from the description which follows,particularly when read in conjunction with the appended figures.

DETAILED DESCRIPTION

Embodiments include hydrogel microparticles for use in biochemical orchemical assays, methods of producing the microparticles, and methods ofperforming biochemical or chemical assays using the microparticles. Eachhydrogel microparticle has a probe region including one or moremolecular recognition elements and an encoded region. The encoded regionincludes multiple portions, with each portion including an associatedplurality of upconversion nanocrystals (UCNs) with a distinct spectralsignature. The multiple portions of the encoding region enable spatialencoding of the microparticle. The associated plurality of UCN for eachregion are selected from a set of spectrally distinguishable UCN, whichenables spectral encoding for each portion of the microparticle. Bycombining spatial and spectral encoding, the microparticles have massivemultiplexing capabilities with superior scaling capability.

The coding scales exponentially as C^(S) for asymmetric particles and asC^(S)/2 for symmetric particles, where C is the number ofdistinguishable spectral signatures (UCN ‘colors’) and S is the numberof spatial features (e.g., microparticle ‘stripes’). For example, for asymmetric microparticle with S encoding portions and a set of Cdifferent spectrally distinguishable UCNs, the following equation liststhe number of codes or unique identifiers that would be available:

$\sum\limits_{x = 0}^{S - 1}C^{({S - x})}$

For example, about 20,000 unique identifiers/codes can be generated fora system in which the encoding region of symmetric microparticles hassix portions and each portion includes a plurality of UCN selected froma set of five different types of spectrally distinct UCNs. As anotherexample, about 500,000 unique identifiers/codes can be generated for asystem in which the encoding region of the symmetric microparticle hassix portions and each portion includes a plurality of nanocrystalsselected from a set of nine different types of spectrally distinctnanocrystals. Thus, a modest number of colors may be coupled with asimilarly modest number of stripes to yield considerable encodingcapacities that scale rapidly with incremental changes to eitherquantity. To increase the encoding capacity, asymmetric microparticlescould be employed. For example, an asymmetric microparticle with sixportions with each portion including one of nine different types ofspectrally distinct nanocrystals would produce over a million uniqueidentifiers/codes.

Some embodiments combine spatial patterning with rare-earth upconversionnanocrystals (UCNs), single wavelength near-infrared excitation andportable charge-coupled device (CCD)-based decoding to distinguishparticles synthesized by means of flow lithography. Some embodimentsexhibit large, exponentially scalable encoding capacities (>10⁶), anultralow decoding false-alarm rate (<10⁻⁹), the ability to manipulateparticles by applying magnetic fields, and dramatic insensitivity toboth particle chemistry.

Some embodiments employ a robust encoding method for compatibility withhigh-throughput particle synthesis and portable CCD-based decoding. Insome embodiments, the resulting particles and decoding system exhibitdramatic insensitivity to particle chemistry—enabling tuning of encodingcapacity and decoding error rate independently of particle materialproperties—as well as the capacity for straightforward magneticmanipulation. In the examples described below, the inventors demonstratequantitatively predictable decoding of both biocompatible particles inchallenging, realistic environments. With single-particle encodingcapacities in excess of 1 million and error rates of less than 1 partper billion (ppb), some embodiments expand the practically accessiblenumber of codes for applications like multiplexed bioassays by orders ofmagnitude.

FIG. 1 schematically depicts an exemplary microparticle 10 for use in abiochemical or chemical assay, in accordance with an embodiment. Themicroparticle 10 has a body 12 including a hydrogel. The body 12includes a probe region 20 and an encoded region 30. The probe region 20includes one or more molecular recognition elements. The encoded region30 includes multiple different portions (e.g., portions 31, 32, 33, 34,34, 35) with each portion (31-35) having an associated plurality ofupconversion nanocrystals (UCNs) (e.g., UCN 41) selected from a set ofspectrally distinguishable UCN (see discussion accompanying FIGS. 2-11below). In some embodiments, one or more portions may not include anynanocrystals and may serve as a “blank” or null portion for encoding. Insome embodiments, the hydrogel body material is mesoporous to allow thediffusion of large (>10 nm) biomolecules though the hydrogel material).

For example, in some embodiments, a first plurality of UCNs with a firstspectral signature is disposed in a first portion 31 of the encodedregion. A second portion 32 of the encoded region includes a secondplurality of UCNs with a second spectral signature different than thefirst spectral signature. In some embodiments, the encoded region of themicroparticle also includes a third portion 33 having a third pluralityof UCNs. In some embodiments, the encoded region of the microparticlealso includes a fourth portion 34 having a fourth plurality of UCNs. Insome embodiments, the encoded region of the microparticle also includesa fifth portion 35 having a fifth plurality of UCNs. The plurality ofmicroparticles in each portion (31-35) of the encoded region is selectedfrom a set of spectrally distinguishable UCNs.

One of ordinary skill in the art in view of the present disclosure wouldrecognize that each microparticle may include an encoding region withfewer than five portions and associated pluralities of UCNs (e.g., fourportions, three portions, two portions) or more than five portions andassociated pluralities of UCNs (e.g., six portions, seven portions,eight portions, nine portions, ten portions, etc.).

The spectral signature associated with a plurality of UCN disposed in aportion of the encoded region is also referred to herein as the spectralsignature of the portion of the encoded region. In some embodiments, twoor more portions of the encoded region may have the same spectralsignature. In some embodiments, two or more portions of the encodedregion with the same spectral signature may be adjacent to each other.In some embodiments, any portions of the encoded region with the samespectral signature must be separated from each other by one or moreportions of the encoded region having different spectral signature(s).In some embodiments, each portion of the encoded region must have aspectral signature different from that of every other portion of theencoded region. In some embodiments, one or more portions of the encodedregion do not include UCNs so that the portion or portions is “blank”without a spectral signature.

The spectral signature of a UCN includes information associated with theemission spectrum of the UCN that distinguishes it from another type ofUCN. In some embodiments, the spectral signature of a UCN or of aplurality UCNs of the same type may include the integrated intensity ofemission of one spectral band (or emission in one spectral range) versusanother spectral band (or emission in another spectral range). Aspectral signature or information regarding a spectral signature may bereferred to herein as a spectral code.

FIGS. 2-10 show emission spectra for an example set of nine spectrallydistinguishable types of UCNs, labeled UCN1-UCN9 respectively, whenexcited with near infrared (NIR) light (e.g., 980 nm light from an NIRdiode laser). UCNs in the example set luminesce in multiple narrow bands(e.g., bands less than 70 nm wide at full width half maximum (FWHM)) inthe visible range when exposed to lower frequency (e.g., near infrared(NIR)) light. Specifically, the example set of spectrallydistinguishable UCNs (e.g., UCN1-UCN10) emit in two or more bandscentered around 470 nm (e.g., 445-500 nm), centered around 550 nm (e.g.,520-560 nm), and centered around 650 nm (e.g., 650-670 nm). Forsimplicity, the 445-500 nm band is referred to herein as the blue band,the 520-560 nm band is referred to herein as the green band, and the640-670 nm band is referred to herein as the red band.

One of ordinary skill in the art in view of the present disclosure wouldrecognize that the set of UCN may include fewer than nine (e.g., eight,seven, six, five, four, three, two) or more than nine (e.g., ten, nine,ten, eleven, twelve, etc.) different types of spectrally distinguishableUCNs. Further, one of skill in the art in view of the present disclosurewould recognize that UCNs having different spectra than those shown, andUCNs than emit in different bands than those shown, also fall within thescope of embodiments. For example, FIG. 11 shows an emission spectrumfor a UCN labeled UCN10 that may be used in the set as an alternative toany of UCN1-UCN9, or in addition to UCN1-UCN9. To augment encodingcapacity, the palette of spectrally distinct UCNs may be furtherexpanded by adjusting Yb—Er—Tm ratios with negligible impact on thedecoding error rate.

The spectral signature of a plurality of UCNs may include informationrelated to the ratio or ratios of the integrated intensities emitted invarious bands (e.g., the ratio of the red band to the green band or viceversa, the ratio of the red band to the blue band or vice versa, theratio of the blue band to the green band or vice versa, or anycombination of the aforementioned). These ratios can be defined withrespect to the emission spectra of the UCNs. However, in someembodiments, the spectral signature of a plurality of UCNs may includeboth information regarding the intensity of light emitted in variousbands and include information regarding the responsivity of the imagesensor to be used. Any detector, image sensor, or imaging device may beemployed. For example, the detector or imaging device may be acharge-coupled device (CCD), a photomultiplier tube-based device (PMT),a complementary metal-oxide-semiconductor (CMOS) imaging sensor, anavalanche photodiode array (APD) imaging device, etc In someembodiments, an imaging sensor with more than one color channel may beemployed.

FIG. 12 shows the spectral responsivity of red 61, green 62 and blue 63channels for a typical RGB CCD device that may be used as a detector insome embodiments. As shown, the red 71, green 72, and blue 73 emissionbands of the exemplary set of UCNs overlap the spectral responsivitiesof the respective red 61, green 62, and blue 63 channel responsivitycurves. For example, FIG. 13 shows the emission spectrum of UCN6overlaying the spectral responsivity of channels of a typical RGBdevice. A convolution of the emission spectrum with the expectedspectral responsivity for each image sensor channel yields curvescorresponding to the expected spectral response of each channel of theCCD image sensor to each type of UCN. The spectral signature for a typeof UCNs can include information regarding the expected spectral responseof an image sensor to a specific UCN emission spectrum, such as a ratioof the expected integrated intensity detected for two color channels.

For example, the Table 1 below shows the expected spectral response of aCCD device to the emission spectra of the UCN3-UCN7 and UCN10 types ofUCNs (see FIGS. 4-8 and 11 for emission spectra). The expected spectralresponse is a convolution of the emission spectrum for type of UCN withthe image sensor channel spectral responsivity shown in FIG. 12.Specifically, Table 1 shows the expected integrated total intensity foreach color channel due to emission of the UCNs. Table 1 also includesratios for the expected total intensity for the green channel to the redchannel, for the blue channel to the red channel, and for the bluecannel to the green channel. Expressing the integrated intensities asratios for different color channels reduces or eliminates the need forcalibration to determine the absolute intensity for any particular colorchannel or emission band.

TABLE 1 Expected Expected Expected Integrated Integrated IntegratedChannel Channel Channel Intensity* Intensity Intensity Ratio Ratio RatioType R Channel G Channel B Channel G/R B/R B/G UCN3 163.4 86.3 0 0.528 00 UCN4 225.4 197.5 0 0.876 0 0 UCN5 91.9 164.5 0 1.790 0 0 UCN7 24.752.1 219.9 2.109 8.9 4.220 UCN6 138.5 158.1 120.4 1.141 0.869 0.7609UCN10 161.6 131.5 0 0.814 0 0

The inventors have found that employing UCN for identifying each encodedregion of a particle has many benefits when compared with othertechniques currently used for encoding particles. For example, someother techniques employ one-dimensional or two-dimensional thicknessvariations or holes in a fluorescently labeled coded region of amicroparticle for identification.

In contrast with UCNs having multiple narrow emission bands, commonlyused fluorescent labeling molecules (e.g., fluorophores) each tend toemit in a single broad band (e.g., DAPI fluorescent dye has a singleemission band that is about 100 nm wide FWHM). In microparticles usingfluorophores for encoding, the broad emission bands of the fluorophoreslimits the number of different fluorophores that may be employed withouthaving significant overlap between emission bands and resultingambiguity in identification. In addition, the absence of multipleemission bands for a single fluorophore may require the use of anexternal calibration standard. In contrast, UCNs have multiple narrowemission bands in different portions of the visible spectrum (e.g.,separated by tens to hundreds of nm). The ratio of intensity of emissionin various bands can be used to distinguish between different UCNs, andalso acts as an internal calibration standard, obviating the need forexternal calibration.

Microparticles using UCNs for encoding may experience less reduction ofthe signal to noise ratio due to autoluminescence than microparticlesusing fluorophores for encoding. Luminescent UCNs absorb light in onerange of wavelengths and emit light in a shorter range of wavelengths(e.g., absorb in the NIR range and emit in the visible range). Incontrast, commonly used fluorophores and quantum dots usually absorblight in a wavelength range and emit light in a longer wavelength range(e.g., absorbing in the ultraviolet range and emitting in the visiblerange). For example, the commonly used fluorophore4′,6-diamidino-2-phenylindole (DAPI) has absorption maximum around 370nm (UV) and an emission maximum around 450 nm (blue). Illumination ofthe fluorophores for identification (e.g., with UV light) may result inunintended autofluorescence of materials and solvents in the visiblewavelengths that decreases the signal to noise ratio, which can be asignificant problem with biological samples. Because the UCNs describedherein are upconverting, the NIR light used to excite the UCNs generallydoes not cause autoluminescence in the shorter wavelengths of thevisible range. Thus, the use of UCN may improve the signal to noiseratio for an encoded region.

Microparticles using different types of UCNs for encoding may requireonly a single narrow band excitation source as opposed to microparticlesusing different types of fluorophores, which may require multiple lightsources to provide excitation in different wavelength bands. Forexample, a 980 nm light source with a power density of less than 10W/cm² (e.g., an near infra-red (NIR) laser diode) may be used as asingle excitation source for multiple different types of UCNs. Incontrast, microparticles using common fluorophores for parts of thevisual light spectrum, such as DAPI (blue), Oregon green 500 (green) andALEXA FLUOR 633 (red) with absorption maximums at 350 nm, 503 nm and 632nm, respectively, may require multiple different excitation sources suchas a UV laser, an argon-ion laser, and a red helium-neon laser.

In some embodiments, the UCNs are rare-earth nanocrystals, which arebright anti-Stokes emitters with tunable spectral properties. IndividualUCNs absorb continuous-wave (CW) NIR light at a single wavelength andemit in multiple narrow bands of the visible spectrum. Large anti-Stokesshifts reduce spectral interference from sample autogluorescence andlead to enhanced signal-to-noise ratios. In contrast to M-ink (anoptically active dye in which nanostructured magnetic materials reflectdifferent wavelengths of light) or quantum dots, these benefits persisteven in the presence of obscurants or a complex reflective background.Tuning of emission intensities in multiple bands by adjusting relativestoichiometries of lanthanide dopants permits ratiometrically uniquespectral encoding, in which the ratio of integrated intensities in twoor more bands serve as the code, rather than absolute intensity. In someembodiments, external spectral standards (e.g., as required by poroussilicon crystals), precise dye loading (e.g., as used with quantum dotsand luminex), sensitive instrumentations (e.g. as required by M-Ink),and extensive calibration may be unnecessary for readout, enabling theuse of standard CCD imaging for decoding.

Example Synthesis of UCNs

Lanthanide-doped NaYF₄ UCNs were made via a scalable batch hydrothermalsynthesis, which is only one of numerous known protocols for synthesisof NaYF₄ UCNs.

Aqueous rare-earth chloride salts, sodium hydroxide, ammonium fluoride,ethanol and oleic acid were heated in a TEFLON-coated stainless steelpressure vessel. Specifically, 2 ml of ReCl₃ (0.4 M, RE=Y, Yb, Er, Gd,Tm) and 2 ml of NH₄F (2 M) were added to a mixture of 3 ml of NaOH (0.6M), 10 ml of ethanol and 10 ml of oleic acid. The solution wastransferred to a 50 ml TEFLON-lined autoclave and heated at 200° C. for2 hours. The resulting products were centrifuged to collect the UCNs,which were then repeatedly washed with ethanol and deionized water andthen re-dispersed in cyclohexane.

During synthesis, the inventors used the concentration of variouslanthanide dopants and the reaction time and temperature to improve theluminescence intensity of the UCNs and to alter the upconversionspectrum of the nanocrystals.

The synthesis procedure described above can produce NaYF₄ UCNs in twodifferent phases having different crystal structures: an α-phase with acubic crystal structure and a β-phase with a hexagonal crystalstructure. Generally speaking, luminescence intensity is significantlyhigher in β-phase crystals than in α-phase crystals due to the lowerratio of surface defects to crystal volume in the β-phase. Without highlevels of gadolinium doping, relatively high temperatures must bemaintained for relatively long times (e.g., 350° C. for 24 hours) toinduce the α→β phase transition in the UCNs. In contrast, the inventorsdoped with 30 mol % gadolinium (Gd) to induce the α→β phase transitionat a lower temperature (200° C.) held for a shorter time (2 hours). TheGd has little to no effect on the shape of the upconversion emissionspectrum generated due to the presence of the other dopants.

Increasing reaction time and increasing reaction temperature tended toincrease the luminescence intensity of the UCNs due to increasednanocrystal size. Increasing the UCN size decreases the ratio of surfacearea to volume for the UCNs, thereby decreasing the ratio of surfacedefects to crystal volume. Further, luminescence for larger UCNs wasless likely to be red-shifted due to preferential quenching of highfrequency emission, which can occur in smaller UCNs.

The concentrations of dopants other than Gd were used to change theupconversion emission spectrum. Spectrally distinct UCNs were producedby adjusting the relative stoichiometries of the lanthanide ions Yb³⁺,Er³⁺ and Tm³⁺ in the UCN reaction premix. The lanthanide dopantstoichiometries have relatively little impact on the UCN nanostructureand surface chemistry, decoupling control of the emission spectrum fromthe particle chemistry and resulting material properties. Ytterbium(Yb³⁺) is an important dopant for bright multicolor emission, because itacts as a high-NIR absorption cross-section and energy transfer agentfor upconverting emission. Increasing the Yb percentage tends to‘red-shift’ the upconversion spectrum, increasing the ratio of theemission intensity in the red band (640-670 nm) relative to the emissionintensity in the green band (520-560 nm) in Erbium (Er³⁺) co-dopedcrystals. FIG. 14 illustrates how increasing the Yb concentration shiftsthe emission spectrum and shifts overall emission color from green toorange. Doping with Er³⁺ at low levels (2% or less) leads to narrowpeaks centered at 550 nm and 650 nm. Overall perceived emission colorfor materials doped with Yb³⁺ and Er³⁺ can range from green to red,depending on the Yb concentration. Doping with Thulium (Tm³⁺) at verylow levels (˜0.2%) leads to emission in the blue band (445-500 nm) and amore intense peak at 800 nm.

The inventors produced ten different types of spectrally distinguishablelanthanide-doped NaYF₄ UCNs labeled UCN1-UCN10, whose spectra appear inFIGS. 2-11. The overall colors of the UCN1-UCN9 types when irradiatedwith an NIR laser diode are shown in FIG. 15, which includes aluminescence image of suspensions of UCN1-UCN9 in cyclohexane upon 980nm near infra-red (NIR) excitation. As illustrated by FIG. 15, thecolors of the UCNs can be readily distinguished by the naked eye. Thecomposition of the dopant used for each type of UCNs is listed in Table2 below. The Y concentration, which makes up the balance of each dopantconcentration, is in square brackets because it is not an active dopant.

TABLE 2 Description Gd Yb Er Tm [Y of Label (mol %) (mol %) (mol %) (mol%) (mol %)] overall color UCN1 30 69.7 0.1 0.2  [0] Violet UCN2 30 69.90.1 —  [0] Red UCN3 30 68 2 —  [0] Orange UCN10 30 40 2 — [28] DarkYellow UCN4 30 30 2 — [38] Yellow UCN5 30 18 2 — [50] Green UCN6 30 200.1 0.2   [49.7] Cobalt UCN7 30 18 — 0.2   [51.8] Blue UCN8 30 18 0.030.2   [51.77] Sky Blue UCN9 30 31.7 0.1 0.2 [38] Grey

FIG. 16 shows transmission electron microscopy (TEM) images of theUCN1-UCN9 types of UCNs produced by the process described above, as wellas an enlarged image of the UCN6 nanocrystals. In FIG. 16, the scalebars are 100 nm. The TEM samples were prepared by placing a drop of UCNsin cyclohexane onto the surface of a copper grid. Overall, the UCNsproduced were rod-shaped with an average size of 250-450 nm in lengthand 40-60 nm in width.

The inventors made several different batches of the same type of UCNs toconfirm that the emission spectra were consistent from batch to batch.Upconversion luminescence spectra of UCNs were measured in a poly(urethane acrylate) (PUA) prepolymer solution (9/1 PUA/PI (v/v)) with afluorescence spectrometer with a 1 W CW diode laser (980 nm) used as theexcitation source. FIG. 17 shows the normalized emission spectra forthree different batches of UCN7 type nanocrystal. As shown, emissionspectra for the three different batches are practicallyindistinguishable on the combined graph.

The high Gd content of UCN1-UCN10 makes the UCNs paramagnetic andsubject to physical manipulation through external magnetic fields. Theinventors confirmed this by manipulating the nanocrystals suspended invials using external ferromagnets. FIG. 18 includes luminescence imagesof UCNs in liquid in a vial (a) settled to the bottom of the vial withno applied magnetic field, and (b) with an applied magnetic field from aferromagnet drawing the UCNs to the left side of the vial. FIG. 19 is agraph of data for magnetization as a function of applied magnetic fieldfor UCN4, which was obtained using a superconducting quantuminterference device (SQUID).

Example Surface Modifications of UCN

The synthesis process described above produced UCNs capped with oleicacid, a fatty acid with a 17-carbon hydrocarbon tail. As a result of theoleic acid capping, the resulting UCNs were insoluble in aqueous media,which created problems with dispersing the UCNs in aqueous orhydrophilic source materials. Furthermore, the UCNs with oleic acidtails luminesced brightly only in hydrophobic media. Exposure of theoleic acid capped UCNs to water caused significant aggregation and ahigh degree of reversible luminescence attenuation due to surfacedefect-mediated quenching.

The inventors utilized a method of modifying the oleic acid tail on theUCNs to improve their solubility in water and increase theirluminescence in hydrophilic media. The oleic acid double bond wasoxidized to form an alcohol, and then cleaved, thereby releasing theoutward-facing hydrophobic part of the oleic acid chain and forming acarboxylic acid group.

The specific procedure employed to modify the oleic acid tail of theUCNs involved adding 0.1 gram of UCNs to a mixture of cyclohexane (100mL), tert-butanol (70 mL), water (10 mL) and 5 wt % K₂CO₃ solution (5mL) and stirring for about 20 minutes at room temperature. Then, 20 mLof Lemieux-von Rudloff reagent (5.7 mM KMnO₄ and 0.1 M NaIO₄ aqueoussolution) was added dropwise to the solution. The resulting mixture wasstirred for 48 hours. The product was centrifuged and washed withdeionized water, acetone, and ethanol. Subsequently, the UCNs weredispersed in hydrochloric acid (50 mL) of pH 4, and stirred for 1 hourforming carboxyl-terminated UCNs, which were washed 5 times withdeionized water and collected by centrifugation. The resultingcarboxyl-terminated UCNs dispersed without aggregation in aqueous mediaand luminesced strongly in hydrophilic media.

The inventors developed a method for modifying the carboxyl-terminatedUCN to form acrylate-terminated UCN that could be cross-linked with thehydrogel material of the microparticle. The method included mixing 200μl of EDC (20 mg/ml) and 200 μl of sulfo-N-hydroxysuccinimide(sulfo-NHS) (20 mg/ml) with 200 μl of carboxy-terminated UCNs in2-(N-morpholino) ethanesufonic acid (MES) buffer (0.1 M, pH 6.0, 40mg/ml) and stirring for two hours at room temperature to activate thesurface as carboxylic acid groups. The NHS-activated UCNs werecentrifuged and washed with water. The precipitate was re-dispersed in200 μl of PBS buffer (0.1 M, 5 ml, pH 7.2) containing 200 μl of2-hydroxyethylacrylate (20 mg/ml). The mixture was then stirred for 24hours at room temperature. The resulting acrylated UCN were purified byrepeated centrifugation (3000 rpm, 5 min, 5 times) and resuspended indeionized water.

FIG. 20 is a flow diagram 110 of a method of making a hydrogelmicroparticle for use in a biochemical or chemical assay. A firstencoded region source material is provided (112). The first encodedregion source material includes a hydrogel and a first plurality of UCNshaving a first spectral signature. For example, the first plurality ofUCNs may be the nanocrystals described above and labeled UCN3. Thespectral signature of the first plurality of the UCNs (type UCN3) may bedescribed as the spectrum shown in FIG. 4, or may be described by theratio of the integrated intensity in one detection channel relative toanother detection channel (e.g., the ratio of the green detectionchannel integrated intensity the red detection channel integratedintensity as shown in Table 1), or by multiple different integratedintensity ratios (e.g., green to red, blue to red, red to green). Asecond encoded region source material is also provided (114). The secondencoded region source material includes a second plurality of UCNshaving a second spectral signature different than the first spectralsignature. The second plurality of UCNs may be the nanocrystalsdescribed above and labeled UCN4. The spectral signature of the secondplurality of the UCN (type UCN4) may be described as the spectrum shownin FIG. 5, or may be described by the ratio of the integrated intensityin one detection channel relative to another detection channel (e.g.,the ratio of the green detection channel integrated intensity the reddetection channel integrated intensity as shown in Table 1), or bymultiple different integrated intensity ratios (e.g., green to red, blueto red, red to green). Although the flow chart only specifies a firstencoded region source material and a second encoded region sourcematerial, the number of encoded region source materials requiredcorresponds to the number of portions of the encoded region desired inthe resulting microparticle. A probe region source material including ahydrogel material is also provided (116).

The first encoded region source material, the second encoded regionsource material, and the probe region source material are cross-linkedforming the first portion of an encoded region 31, the second portion ofthe encoded region 32, and the probe region 20. The probe region 20 iscross-linked with one or both of the first portion 31 and the secondportion 32 of the encoding region to form a contiguous microparticle. Inembodiments with more than two portions of the encoded region, eachportion is cross-linked with one or more other portions of the encodedregion and/or with the probe region.

In some embodiments, the UCNs for at least some of the portions of theencoded region have a hydrophilic surface. In some embodiments, the UCNsfor at least some of the portions of the encoded region have ahydrophilic ligand. In some embodiments, providing the first encodedregion source material and providing the second encoded region sourcematerial may include modifying the first plurality of nanocrystals andthe second plurality of nanocrystals to have a hydrophilic surfaceand/or a hydrophilic ligand. Having a hydrophilic surface and/or ahydrophilic ligand may aid in dispersing the UCNs in the respectivesource material.

In some embodiments, the UCNs for at least some of the portions of theencoded region have acrylated ligands for cross-linking with thepolymers of the hydrogel matrix. In some embodiments, providing thefirst encoded region source material and providing the second encodedregion source material may include modifying the first plurality ofnanocrystals and the second plurality of nanocrystals to includeacrylated ligands. In some embodiments, the plurality of UCNs is boundto the polymer material at the time of particle synthesis through anacrylate group.

In other embodiments, another type of covalent linkage could be madebetween the UCNs and the hydrogel matrix. The UCNs can be bound to thehydrogel matrix using any number of covalent attachment mechanisms(e.g., amide linkages, disulfides, esters, ethers, aldehydes/ketones,cycloadditions, click chemistry, azides, and carbamates).

In some embodiments, at least some of the UCNs are doped with rare-earthmetals. In some embodiments, at least some of the UCNs are doped with acomposition including at least 30 mol % Gd. In some embodiments, atleast some of the UCNs are paramagnetic.

In some embodiments, the material for each portion of the encoded regionand for the probe region is the same material. In some embodiments, thematerial for the portions of the encoded region is different than thematerial for the probe region.

As noted above, in some embodiments, the UCNs have a hydrophilicsurface. In some embodiments, the UCNs have a hydrophilic ligand. Havinga hydrophilic surface and/or a hydrophilic ligand may aid in dispersingthe UCNs in the source material.

In some embodiments, the method also includes co-flowing the sourcematerial for each encoded region and the source material for the proberegion to an area for cross-linking. For example, a stop-flowlithography (SFL) technique may be employed for forming themicroparticles. In SFL, viscous UV-sensitive pre-polymer solutions(which may be referred to herein as source materials) undergo laminarco-flow into a small microfluidic device, which may be made ofpolydimethylsiloxane (PDMS). For organic synthesis, the microfluidicdevice may be made from perfluoropolyether (PFPE). The flow of thepre-polymer solutions is stopped for a brief period in which thepre-polymer solutions in the device are exposed to photomask-patternedultraviolet light. The UV light causes cross-linking, polymerization, orboth within milliseconds in the region delineated by the photomaskforming micro-sized polymeric particles. The shape of each particle isdefined by the photomask. The composition of each striped portion of theparticle is determined by the composition of the laminar co-flowingstreams (e.g., the source materials). The SFL technique is particularlywell suited for spatial and spectral encoding of microparticles usingnanocrystals because of the ability to control both overallmicroparticle particle shape and the composition of different stripedportions of the microparticle.

FIG. 15 schematically depicts SFL being used to make a hydrogelmicroparticle with a probe region and an encoding region with differentportions of the encoding region including UCNs with distinguishablespectral signatures. In the diagram the encoded region source materialsare labeled ERSM1-ESRM5, and the probe region source material is labeledPRSM. Each of the encoded region source materials includes a pre-polymer142 and a plurality of UCNs, which may be acrylated UCN 144 in someembodiments. As used herein, the term pre-polymer includes monomers, andpolymer chains that can be cross-linked. As used herein, the termcross-linking refers broadly to forming links between polymer chains, toforming links between a polymer and a nanoparticle, and topolymerization of monomers. The one or more encoded region sourcematerials ERSM1-ERSM5 and one or more probe region source materials PRSMare flowed to an area 150 within a microfluidic device. When theco-flows are briefly stopped, a light source 160 (e.g., a 350 nm UVlight source) a photomask 162 and a focusing optic (e.g., objective lens164) provide patterned and focused light at the area 150 forcross-linking/polymerization of the pre-polymer 142. Cross-linking 146of the pre-polymer source materials forms the microparticle 170 bycreating a hydrogel polymer network. As shown, the UCNs 144 may includeacrylated ligands, which allows the UCNs 144 to crosslink 146 with thehydrogel polymer network 148. Each encoded region source materialERSM1-ERSM5 forms a corresponding portion 171-175 of the encoded regionand the probe region source material PRSM forms the probe region 180 ofthe microparticle 170. In some embodiments, the UCNs are notcross-linked with the hydrogel polymer network, but instead arephysically entrained by the matrix pore size of the hydrogel polymernetwork.

Although photomask 162 is shown having a pattern that only forms onemicroparticle at a time, in some embodiments, the photomask may have apattern for forming multiple microparticles simultaneously. In someembodiments, a photomask may have a pattern that produces microparticleshaving different shapes simultaneously. In some embodiments, thephotomask may produce asymmetric particles and/or particles havingnonrectangular shapes.

Although microparticle 170 is shown with five encoded regions, in otherembodiments, there may be more or fewer than six encoded regions. Forexample, FIG. 22 shows luminescence images of various microparticleseach having between two to six encoded regions. Microparticles with anadditional encoding region would boost the encoding capacity whilerequiring little more than an additional input port on the microfluidicsynthesis device.

For further details regarding the SFL technique for forming hydrogelmicroparticles, see U.S. Patent Application Publication No. US2012/0316082 A1, published Dec. 13, 2012, and U.S. Patent ApplicationPublication No. US 2012/0003755 A1, published Jan. 5, 2012, each ofwhich is incorporated by reference herein in its entirety. An exemplaryflow lithography system is described below with respect to FIGS. 42 and43.

Example Production of PEG-DA Hydrogel Microparticles with UCNs

The inventors produced polyethylene glycol diacrylate (PEG-DA) polymermicroparticles by stop flow lithography. Initially, the inventors madesets of microparticles, with each set including only one type ofnanocrystal to determine whether incorporating the nanocrystals intomicroparticles changes the emission spectral of the nanocrystals. Foreach of the nanocrystal types UCN1-UCN10, fifty PEG-DA hydrogelmicroparticles were produced. A CCD device was used to obtain a threecolor image (red channel, green channel and blue channel) of eachmicroparticle while illuminated by NIR light producing a red channelimage, a green channel image and a blue channel image. For each channelimage, the intensity (pixel value) within the boundaries of eachmicroparticle was integrated yielding a “pixel value” for each channelfor each microparticle. FIG. 23 includes histograms of the integrated“pixel values” for the red, green and blue channels from fiftymicroparticles for the UCN1-UCN9 types. The histograms for some of thetypes also include an inset image of a representative NIR-illuminatedmicroparticle. As shown by the inset images, a stop flow lithographyprocess can be used to make different microparticle shapes.

The mean measured integrated intensity values from fifty microparticlesfor each type of UCNs were then compared with the expected integratedintensity data obtained from a convolution of the UCN emission data andthe image sensor response curves. Table 3 below includes measured meanintegrated intensity data, the standard deviation and the coefficient ofvariability for UCNs in microparticles. Expected integrated intensitydata based on emission spectra from UCNs in solution are also includedfor comparison. As shown in the table, the mean integrated intensity andthe expected integrated intensity values are consistent. The averagecoefficient of variation across all particles and UCN colors was 2%.This corresponds to an average standard deviation of 2.1 RGB units (on ascale of 255) for separately acquired images of separately synthesizedparticles, indicating outstanding particle-to-particle reproducibility.In addition, error ellipses are non-overlapping to better than 6 sigma,indicating that decoding error rates of less than 1 ppb are to beexpected. Thus, if the emission spectrum of a type of nanocrystals isknown, the integrated intensity for detection in a color channel can bereliably predicted.

TABLE 3 Mean Expected Mean Integrated Integrated Integrated ExpectedIntensity ± Expected Mean Intensity ± Intensity ± Integrated standardIntegrated Integrated standard standard Type Intensity deviation CvIntensity Intensity Cv deviation deviation Channel R R R G G G B B CvUCN1 130.3 126.34 ± 1.43 0.02 68.5  65.30 ± 2.29 0.03 103.7 100.74 ±2.48 0.02 UCN2 103.3 109.10 ± 1.87 0.01 44.8  42.70 ± 1.39 0.03 10.2 17.37 ± 1.43 0.08 UCN3 164.5 164.29 ± 2.26 0.01 91.9  91.73 ± 2.73 0.020 0 — UCN10 161.6 160.86 ± 1.3 131.5 130.97 ± 1.3 0 0 — UCN4 225.4225.89 ± 2.29 0.01 197.5 194.71 ± 2.01 0.01 0 0 — UCN5 91.9  86.10 ±1.42 0.01 164.5 161.77 ± 1.89 0.01 0 0 — UCN6 120.4 123.52 ± 2.15 0.01158.1 163.40 ± 2.04 0.01 138.5 132.29 ± 2.54 0.02 UCN7 24.7  23.54 ±2.02 0.08 55.1  63.22 ± 1.93 0.03 219.9 222.36 ± 2.9 0.01 UCN8 83.2 78.37 ± 2.59 0.01 132.6 128.58 ± 2.63 0.02 182.2 189.61 ± 1.89 0.01UCN9 158.9 151.34 ± 2.02 0.01 131.1 127.62 ± 1.93 0.02 120.6 125.73 ±2.92 0.02

FIG. 24 is a scatterplot showing the red channel, green channel, andblue channel integrated intensity values for each microparticlesincorporating the UCN1-UCN9 type nanocrystals. All of the UCN1-UCN9types of nanocrystals have red channel and green channel emissionintensities. The UCN1, UCN2, UCN6, UCN7, UCN8 and UCN9 types ofnanocrystals have emission intensities in the blue channel as well asthe red and green channels. The ellipses around each cluster of datapoints are the three-sigma, four-sigma and five-sigma contours derivedfrom fitting a Gaussian mixture model to the data. As shown byseparation between the tight clusters, the UCN type for eachmicroparticle can clearly be distinguished using the red channel, greenchannel, and blue channel integrated intensities for the microparticle.FIG. 25 shows a comparison of the mean integrated intensity value(measured value squares) and the expected integrated intensity value(convoluted value circles) in the green channel versus the red channelfor particles integrating UCN1-UCN9 types of nanocrystals. The ellipsesrepresent the five-sigma confidence contours.

Thus, the inventors demonstrated noise-robust spectral discrimination ofsix different types of UCNs in hydrogel particles illuminated using anNIR diode laser and imaged using a standard CCD camera. Further, asshown by the green channel vs. red channel plot, the red channelintegrated intensity and the green channel integrated intensity aresufficient to distinguish between the six different types ofnanocrystals. The FIGS. 24 and 25 scatter plots reveal that clusteroverlap occurs only past six standard deviations from the mean, implyingan expected error rate of less than 1 part per billion (ppb).

The inventors also compared different batches of hydrogel microparticlesproduced at different times to determine the reliability and thepredictability of the integrated intensities of microparticles fromdifferent batches. Five separate batches of fifty microparticles wereproduced, each batch including the same UCN4 type nanocrystals. Themicroparticles were illuminated with an NIR light source and colorimages were obtained using a CCD camera. Integrated intensity data wasgenerated for microparticles in all five batches and the averageintegrated intensity values for each batch were compared. FIG. 26 is agraph comparing the average integrated intensities for the green channeland for the red channel for each batch of fifty microparticles. Theintegrated intensities in the red and green channels were consistentacross the five batches. As expected, there was no detected signal theblue channel. Table 4 below lists the measured red and green channelintegrated intensity values for each batch showing the consistency andreproducibility of the spectral signature for different batches ofmicroparticles.

TABLE 4 Red Channel Green Channel Mean Integrated Intensity ± MeanIntegrated Intensity ± Type standard deviation standard deviation 1225.89 ± 2.29 194.71 ± 2.01 2 226.51 ± 2.97 195.46 ± 3.14 3 226.35 ±3.42 195.36 ± 3.34 4 226.36 ± 3.01 194.22 ± 2.46 5 224.65 ± 2.05 194.68± 2.77

The inventors confirmed that the oxidation and acrylation process doesnot change an emission spectrum of the UCNs. FIG. 27 is a graph ofemission spectra of UCN4 type nanocrystals after each step in thesurface chemical modification of the UCNs (e.g., before processing incyclohexane, after oxidation, after acrylation, and in PUA prepolymersolution). The spectra overlay each other establishing that surfacechemistry modifications of the UCNs before incorporation intomicroparticles does not significantly affect emission spectra of theresulting particles.

The inventors also confirmed that there was no attenuation of theluminescence response of the UCNs integrated into hydrogelmicroparticles upon prolonged intense NIR irradiation due tophotobleaching. FIG. 28 is a graph of intensity as a function of timefor hydrogel microparticles including UCN7 type nanocrystals uponcontinuous exposure to a 980 nm NIR light from a 1 W laser. This is incontrast to many commonly used fluorophores which exhibit attenuationdue to photobleaching.

The inventors also compared the stability of hydrogel microparticlesmade with carboxyl-terminated UCNs, in which the nanocrystals aretrapped in pores in the hydrogel matrix, and hydrogel particles madewith acrylated UCNs, in which the nanocrystals are bonded to thehydrogel matrix via acrylates. FIG. 29 includes graphs comparingintensity as a function of age of microparticles including acrylatedUCN7 type nanocrystals and microparticles including carboxyl-terminatedUCN7 type nanocrystals without acrylation. As shown, there is areduction in emission intensity of the microparticles includingcarboxyl-terminated UCNs without acrylation over 30 days, presumably dueto the UCNs diffusing out of the microparticles. In contrast, themicroparticles with acrylated UCNs showed no attenuation over 30 days ofaging. Thus, acrylation of the UCNs and subsequent bonding to thehydrogel matrix improves the luminescence stability (e.g., theshelf-life) of the microparticles.

Example Formation of PEG-DA Hydrogel Microparticles with Spectral andSpatial Encoding

After establishing the predictability and reproducibility of the methodfor forming UCNs and the predictability and reproducibility of thespectra from hydrogel particles that each include only one type of UCNs,the inventors produced PEG-DA hydrogel microparticles with both spectraland spatial encoding. PEG-DA microparticles are biocompatible andmesoporous allowing diffusion of large biological macromolecules. Stableintegration of UCNs into microparticles involved use of hydrophilicsurface chemistry with a UV-active functional group on the UCNs forstrong, covalent incorporation as described above. In an embodiment inwhich the hydrogel is more densely cross-linked covalent incorporationof the UCNs may not be needed. In an embodiment in which the UCNs aredisposed in a non-hydrogel portion of the particle, covalentincorporation of the UCNs may not be needed. For example, inmicroparticles with a hydrogel probe region and a PUA encoded region,the dense cross-linking of the PUA and the hydrophobic surface chemistrylarge, rod-like UCN nanostructure may enabled homogeneous andirreversible physical entrainment of the UCNs in the PUA portion.

Specifically, elongated hydrogel microparticles were produced that eachincluded a probe region and an encoding region. The encoding region wasdivided into five portions, (e.g., five stripes) with each portionincluding a plurality of UCNs having distinguishable spectral signature.Although the microparticles produced included five portions of a encodedregion, in some embodiments, each microparticle may have an encodedregion with more than five portions or less than five portions. Althoughthe hydrogel microparticles produced were rectangular and elongated, insome embodiments, the hydrogel microparticles may have a differentaspect ratio and/or a different shape. Further, the microparticlesproduced may be symmetric or asymmetric.

The microparticles were produced by SFL using encoding region sourcematerials and a probe region source material. Specifically, for eachencoding region source material, acrylated UCN were dispersed in aPEG-DA premixture solution yielding a mixture of 45 vol % PEG-DA(Mn=700), 40 vol % UCNs (0.5 mg/μl) 10 vol % poly(styrenesulfonate) PSSand 5 vol % DAROCUR 1173 photoinitiator (PI)). For microparticles with aPUA encoded portion, the PUA microparticle source material comprised 150mg of UCNs dispersed in 300 μl of a 9:1 volume ratio PUA/PI solution.

The probe region source material employed a similar PEG-DA premixturesolution that also included molecular recognition element, specificallya nucleic acid probe for miRNA target molecules. The source materialswere used to form microparticles using SFL as described above withrespect to FIG. 21.

A microfluidic device was fabricated from poly-dimethylsiloxane (PDMS)for the SFL system. PDMS was mixed with a curing agent in a 10:1 ratioand degassed under vacuum for 30 min. Degassed PDMS was poured onto anSU-8 master mold and cured overnight at 65° C. Channels were then cutout of the mold and bonded with a glass slide coated withpartially-cured PDMS in order to assure oxygen permeability. Theassembled device was fully cured overnight at 65° C. The microfluidicchannel in the microfluidic device of the SFL system was 300 μm wide and36 μm high.

A photomask for the SFL was designed using a computer added draftingprogram and printed with a high-resolution printer. The mask was placedin the field-stop of a microscope before synthesis. A microfluidicdevice was fabricated from poly-dimethylsiloxane (PDMS) for the SFLsystem. PDMS was mixed with a curing agent in a 10:1 ratio and degassedunder vacuum for 30 min. Degassed PDMS was poured onto an SU-8 mastermold.

The microfluidic channel of the SFL system was loaded with the compositemonomer solution, aligned on a microscope stage, and subjected to apressure-driven flow. In every synthesis cycle, the monomer flow washalted (350 ms) and particles were photo-polymerized in the device usingUV light filtered through a dichroic filter set (365 nm wavelength lightfor 100 ns exposure tine). The polymerized particles were then covectedinto a collection tube for 500 ms. Synthesis occurred at a rate of ˜5particles per second. After synthesis the particles were rinsed. The PEGparticles were rinsed 3 times with 1×TET (1×TE with 0.05% (v/v) Tween20).

Although PEG-DA and PUA were used for the hydrogel microparticles andpartial hydrogel microparticles in the examples described herein, anydi-acrylated monomers that have been used in stop-flow lithography maybe used for the encoded region. Further, any di-acrylated monomers intowhich UCNs (either nanocrystals with modified surfaces or ligands ornanocrystals with unmodified surfaces or ligands) may be well-dispersedcan be employed.

In an initial batch of encoded hydrogel microparticles used for testing,each portion of the encoded region included a plurality of nanocrystalsselected from the set of types UCN3, UCN4, UCN5 and UCN7, whosecharacteristics are described above. As used herein, encodedmicroparticles refers to microparticles that each have one or moreportions of the encoded region and that each have one or more types ofspectrally distinguishable UCNs. Eight encoded microparticles wereilluminated with the NIR diode laser and imaged using a standard CCDimage sensor. The integrated intensity was calculated for the red andgreen channels of the image sensor. FIG. 30 is a plot of the greenchannel integrated intensity vs. the red channel integrated intensityfor each portion of the encoded region in the eight microparticles. Asshown, the integrated intensities for the portions of the encodedregions are clumped into groups corresponding to the UCN3, UCN4, UCN5and UCN7 nanocrystals types. The ellipses are the five-sigma Gaussianfits to the data from the particles having only one type ofnanocrystals, which may be considered the “training data.” All of thedata points for the encoded particles fell within the five-sigmaGaussian fit for the training data.

FIG. 31 schematically depicts detection of a target nucleic acid ofinterest by a molecular recognition element, specifically a nucleic acidprobe 182, in a probe region 180 of a microparticle 170. In someembodiments, the nucleic acid probe 182 includes a capturing sequence182 c for binding a targeted nucleic acid of interest 184 and anadjacent adapter sequence 182 a for binding a universal adaptor. Uponexposure to the target nucleic acid in a sample solution, the targetnucleic acid and the capturing sequence hybridize as indicated by arrowA1. After exposure to the sample, the microparticle is exposed to auniversal adapter 186 (e.g., a biotinylated universal adapter), whichbinds to the adapter sequence of the nucleic acid probe and to thehybridized target nucleic acid as indicated by arrow A2. Themicroparticle is then exposed to a reporter molecule, such as afluorescence reporter (e.g., streptavidin-phycoerythrin (SA-PE)) thatbinds to the universal adapter. For a more detailed explanation andother examples of molecular recognition elements and detectable entitiesthat may be employed in the probe region see U.S. Patent ApplicationPublication No. US 2012/0316082 A1, published Dec. 13, 2012, which isincorporated by reference herein in its entirety.

PEG-DA particles with distinct coding and bioassay regions weresynthesized, each including an encoding region with five encodingregions (i.e., 5 stripes) yielding an encoding capacity on the order of10⁵. One set of the synthesized particles contained a microRNA (MiRNA)probe for miR-210 and another contained a probe for mi-R221.

The inventors produced microparticles having two different codes for usein a multiplexed assay. Microparticles with the first code (UCN4, UCN5,UCN3, UCN7, and UCN4 or 45734) included a probe region with a molecularrecognition element for 210 miRNA (miR-210). Microparticles with thesecond code (47534) included a molecular recognition element for 221miRNA (miR-221). Images of the two encoded microparticles under NIRillumination are shown in FIG. 32.

In order to compare performance of spectrally-encodednanoparticle-containing hydrogel microparticles particles to hydrogelparticles without UCNs particle, a batch of “standard” hydrogelparticles including a miR-221 probe region flanked by two controlregions were synthesized. The standard particles had no encoding regionand no UCNs. Both the encoded hydrogel microparticles and the standardhydrogel microparticles had probe regions of identical dimensions toensure similar mass transport and reaction inside the gel network. FIG.33 shows a bright field image and a fluorescence image of the standardparticle with no encoding region and no UCNs after exposure to miR-221.FIG. 34 shows a graph comparing the fluorescence intensity in the proberegion for microparticles with the second code exposed to miR-221 andfor the control particles exposed to miR-221 including data for sixparticles of each. As shown by FIG. 34, the fluorescence intensity ofthe probe region was not affected by the presence of the encoded regionsof UCNs.

The spectrally-encoded hydrogel particles functionalized with DNAcapture probes for miR-210 and miR-221 were used in a microRNA assay todemonstrate specific and multiplexed detection of the two targets. Inthe multiplexed assay, microparticles with the two different codes wereexposed to four different sample solutions: one containing 500 amol ofmiR-210, one containing 500 amol of miR-221, one containing 500 amol ofboth 221 miR-210 and miR-221, and one including neither. This enabledevaluation of encoded microparticles both with regard to standardparticles and with regard to specificity. Post-target incubation, boundmiRNA targets were labeled using a biotinylated universal linkersequence and a streptavidin-phycoerythrin (SA-PE) fluorophore, andimaged under fluorescence.

Assay reactions were carried out in a final volume of 50 μL inside a0.65 mL Eppendorf tube. Each reaction contained a total of 75 particles(25 particles of each type: (standard miR-221, spectrally-encodedmiR-221, spectrally-encoded miR-210)). Target incubations were carriedout in microRNA hybridization buffer for 90 minutes at 55° C. using athermoshaker (1500 RPM). Post-incubation, particles were rinsed withthree 500 μl volumes of microRNA rinse buffer (RB) using centrifugation.After each rinse, supernatant was manually aspirated, leaving 50 μL ofsolution and particles in the reaction tube. A volume of 235 μL of aligation mastermix, which was prepared using 100 μL 10×NEB2, 900 μL TET,800 U/mL T4 DNA ligase, 40 nM biotinylated universal linker sequence,and 250 nM ATP, was then added to the reaction for a 30 minuteincubation at 21.5° C. and 1500 RPM. Microparticles were rinsed threemore times using microRNA RB and incubated withstreptavidin-phycoerythrin at a final concentration of 2 μg/mL for 45minutes at 21.5° C. and 1500 RPM. After three more rinses with microRNARB, particles were exchanged into PTET (TET with 25% (v/v) PEG-200) forimaging. DNA sequences for the two probes and the universal linkerappear in Table 5 below.

TABLE 5 DNA Sequences Target Probe Sequence SEQ ID miR-2105Acryd/GAT ATA TTT TAT CAG CCG No: 1 CTG TCA CAC GCA CAG/3InvdT SEQ IDmiR-221 5Acryd/GAT ATA TTT TAG AAA CCC No: 2AGC AGA CAA TGT AGC T/3InvdT SEQ ID Universal/5Phos/TAAAATATATAAAAAAAAAAAA/ No: 3 Linker 3Bio/

FIG. 35 shows images of microparticles with the first code andmicroparticles with the second upon exposure to the four differentsample solutions. Fluorescence images and images under NIR illumination(1 W 980 nm NIR diode laser) were captured separately. In the images inFIG. 35, the fluorescence image of each microparticle overlays the imageof the microparticle under NIR illumination. As shown, the assaysuccessfully discriminated between the presence of miR-210 and miR-221in the four sample solutions.

FIG. 36 is a graph of integrated intensity of each portion of eachmicroparticle for the red and green color channels for the encodedPEG-DA microparticles used for the bioassay. As shown, the encodedPEG-DA microparticle data fits within the five-sigma contours of thetraining data for all of the encoded PEG-DA microparticles, which meansthat error rates of less than 1 part per billion (ppb) may be achieved.

FIG. 37 is a graph of integrated intensity of each portion of eachmicroparticle for the red and green color channels for the PEG-DAmicroparticles used for the bioassay and for PUA microparticles used forlabeling of a blister pack. As shown, the data fits within thefive-sigma contours for both types of microparticles. Thus, thereliability of identification of encoded regions applies acrossdifferent microparticle materials.

The composite images shown in FIG. 35 and the data in FIGS. 34 and 36demonstrate successful multiplexed miRNA detection, and that theencoding strategy has negligible impact on the fluorescence intensityobserved in the probe region, which is an important criterion forquantifying biomolecule concentrations.

Although the microparticles produced included a nucleic acid probe formiRNA as a molecular recognition element, one of ordinary skill in theart would recognize that many different types of molecular recognitionelements could be employed and incorporated into the probe region sourcematerial of various embodiments. For example, other types of molecularrecognition elements that could be employed include, but are not limitedto various nucleic acids, miRNA, ssDNA, proteins, receptor proteins,antibodies, enzymes, peptides, aptamer, avimers, Fc domain fragments,phage, carbon nanotube sensors, peptides, etc. Any existing molecularrecognition element, biological or not, compatible with the particlesynthesis process, may be incorporated.

Although the microparticles produced only included one probe region, insome embodiments, each microparticle may include more than one proberegion. For embodiments with more than one probe region, the differentprobe regions may have different types of molecular recognitionelements. In some embodiments, multiple types of molecular recognitionelements may be incorporated into one probe region. Although themicroparticles produced include a probe region that is distinct from theencoded region, in some embodiments, the probe region may partially orcompletely overlap with one or more portions of the encoded region.

Example Formation of Hydrogel Microparticles with PEG-DA Probe Regionand PUA Encoded Region

As noted above, after the inventors selected polyethylene glycoldiacrylate PEG-DA as a suitable biocompatible polymer for forming thehydrogel of the probe region, it was discovered that oleic-acid cappedUCNs do not disperse in the PEG-DA. Instead the oleic-acid capped UCNsaggregated forming clumped distributions in the PEG-DA pre-polymersolution, which led to clumped distributions of UCNs in themicroparticles. Before the inventors developed the method of modifyingthe oleic acid group to form carboxyl-terminated UCNs, the inventorsinitially employed a hydrophobic polymer, polyurethane acrylate (PUA)(specifically MINS-300 produced by Minuta Tech, Co. Ltd. of Gyeonggi-DoKorea) for the encoded region in an attempt to address the problem ofUCN aggregation. The nanoparticles dispersed well in the MINS-300hydrophobic PUA, but the MINS-300 hydrophobic PUA wouldn't form a robustcross-linked interface with the PEG-DA of the probe region. Theinventors then selected another PUA that is only slightly hydrophilic(specifically MINS-0311 produced by Minuta Tech, Co. Ltd. of Gyeonggi-DoKorea) for the encoded region. The UCNs did not disperse as well in theslightly hydrophilic second PUA MINS-0311; however, the MINS-0311 secondPUA formed a robust interface with the PEG-DA of the probe region uponcross-linking. Using the MINS-0311 second PUA as the polymer for theencoded region source materials, the inventors were able to achieve somedispersion of the UCNs in the portions of the encoded region and arobust interface with the PEG-DA probe region.

FIG. 38 is a microscope image of microparticles 210 with a PEG-DA proberegion 220 (outlined in white) and a second PUA (specifically, MINS-311)encoded region 230 excited by a NIR light source. Each portion of theencoded region 231, 232, 233, 234, 235 included a plurality of UCN. Thepresence of color throughout each portion of the encoded region 231-235indicates that the UCNs were distributed throughout the sourcematerials. However, the nonunifomity of the color in each encoded region231-235 indicates that the UCNs were not uniformly distributed in thesource materials. For comparison, see the images of microparticles inFIG. 26. The microparticles 210 formed with a PEG-DA probe region 220and a PUA encoded region 230 experienced deformation due to differentamounts of swelling in aqueous solvents for the two materials as shownby the white outline of the probe region 220. Despite theseshortcomings, the microparticles 210 could still be employed in somebioassay applications. Further, the inventors demonstrated that the UCNcould be integrated into a microparticle that has chemically distinctpolymers in different portions of the particle.

Any di-acrylated monomers that have been used in stop-flow lithographymay be used for the encoded region. Further, any di-acrylated monomersinto which UCN (either UCNs with modified surfaces or ligands or UCNswith unmodified surfaces or ligands) may be well-dispersed can beemployed.

FIG. 39 schematically depicts a method of performing biochemical orchemical assay 310. The method 310 includes exposing a sample to aplurality of microparticles (312). Each microparticle includes ahydrogel or partial-hydrogel body. The body includes a probe region withone or more molecular recognition elements and an encoded region. Thebody also includes a first plurality of UCN disposed in the firstportion of the encoded region and a second plurality of UCN disposed ina second portion of the encoded region spatially separated from thefirst portion of the encoded region. A spectral signature of the secondplurality of UCN is different than a spectral signature of the firstplurality of UCN. The method also includes 314 illuminating eachmicroparticle with an excitation light source (e.g., an NIR lightsource) (314). The method further includes detecting light emitted fromthe illuminated microparticle (316). The detected light includingupconverted luminescent light from the first plurality of UCN and thesecond plurality of UCN and light associated with the one or moremolecular recognition elements. The method also includes identifyingeach microparticle based on the detected light.

A method of reading out the spectral codes of a microparticle isdescribed with respect to FIGS. 40 and 41, which illustrate reading outa microparticle having six encoded regions and no probe region. One ofordinary skill in the art in view of the present disclosure wouldappreciate how the method may be applied for reading out spectral codesof a microparticle having an encoded region with multiple portions and aprobe region. Initially, a maximum or minimum is identified along the xor y axis (step 1). A center and end points of the particle areidentified (step 2). A particle orientation is determined and, in thecase of an asymmetric particle, a direction of the particle isdetermined, and the center of each stripe is identified (step 3). Anaverage RGB value is calculated within a sampled area around each stripe(step 4).

Specifically, images of particles with 6 stripes were taken via a CCDdecoder and loaded into image processing and analysis software (e.g.,MATLAB by Mathworks of Natick, Mass.). Particle boundaries were definedusing a grayscale intensity-based edge detection algorithm. Boundarypixel x and y values were averaged to determine the particle centroid.Boundary pixels with minimum and maximum x and y values (four pointstotal) were noted, and distances between adjacent points used todetermine the particle end point, or the pixel located on the 2ndshortest edge of the particle boundary and its longitudinal axis. Theend pixel and centroid pixel were then used to determine both the codeorientation and a director for the particle's longitudinal axis. Thecentroid of each striped region of the particle was determined bysegmenting the particle into six regions (the number of stripes werepresumed known a priori) along its longitudinal director. In otherembodiments, k-means image segmentation algorithms may be employed todefine regions of the particle based on color, without a prioriknowledge of the number of particle stripes. RGB values were measured byaveraging pixels within each of the six striped regions of particlesunder test were compared against training RGB values and standarddeviations, as determined from a particle training set. If an averageset of RGB values fell within 3.5 standard deviations of a training RGBvalue, the values were determined to match. In this way, ‘analog’ RGBsequences were translated into ‘digital’ sequences of spectralsignatures.

To test the identification, multiple microparticles were generated witha “true code” and some with a different “false code” as shown in FIG.41. An automated decoding system employing the process described abovewith respect to FIG. 40 correctly distinguished the “true code”microparticles that matched a provided “authentic code” from the “falsecode” microparticles that did not match the provided authentic code,using luminescence images. In FIG. 41, the identified “false code”images are indicated with a box around the image.

Further details regarding an exemplary system of particle synthesis areprovided below. FIG. 42 schematically depicts a flow lithography anddecoding system for particle synthesis that includes a flow lithographymicroscope setup, a decoding microscope setup, and a spectrometer setup.FIG. 43 is an image of the flow lithography and decoding system forparticle synthesis. The flow lithography microscope setup includes a UVLED light source, a 10× objective (Edmund optics), a CMOS camera, adichroic cage cube, a dichroic mirror, cage cube-mounted turning prismmirrors, an XYZ sample stage, a mask holder, φ1″ lens tubes, an XYtranslator, a high-precision zoom housing for 01″ optics, a 30 mm cage,posts, an LED and valve control relay, which were controlled withinstrument control hardware and software, a camera adapter, and a CCDcamera. The decoding microscope setup included a 1 W 980 nm laser, a 950nm cut-on filter, a collimator, a CCD camera adapter, and a CCD camera.The spectrometer setup included a spectrometer, a laser translationstage, an X,Y translating lens mount, NIR achromatic doublet pairs, acollimator, a 950 nm cut-on filter, a 30 mm cage, and posts.

The versatile, high-performance stop-flow lithography (SFL) systems andtechniques described herein are a high throughput process forsynthesizing particles. In a semicontinuous process, multiple coflowinglaminar streams—each containing a single optically active UCN moiety orprobe molecule—are convected into a microchannel (e.g., formed frompoly(dimethylsiloxane) (PDMS) or a non-swelling thiolene-based resin foruse with organic solvents), stopped, and photopolymerized in place viamask-patterned ultraviolet light (365 nm) to form barcoded particles ata rate of 18,000 particles/hr, which are then displaced when flowresumes. This ˜10⁴ particles/hr synthesis rate is by no means limiting;hydrodynamic flow focusing has been used to increase the synthesis ratefor similar particles to over 10⁵ particles/hr. The synthesis platformmay also be constructed using commercial off-the-shelf parts andfree-standing optics. Parallelization in an industrial setting, with nofurther optimization, could readily increase the facility-scalesynthesis throughput by orders of magnitude to meet industrial demand.

In describing exemplary embodiments, specific terminology is used forthe sake of clarity. For purposes of description, each specific term isintended to at least include all technical and functional equivalentsthat operate in a similar manner to accomplish a similar purpose.Additionally, in some instances where a particular exemplary embodimentincludes a plurality of system elements, device components or methodsteps, those elements, components or steps may be replaced with a singleelement, component or step. Likewise, a single element, component orstep may be replaced with a plurality of elements, components or stepsthat serve the same purpose. Moreover, while exemplary embodiments havebeen shown and described with references to particular embodimentsthereof, those of ordinary skill in the art will understand that varioussubstitutions and alterations in form and detail may be made thereinwithout departing from the scope of the invention. Further still, otheraspects, functions and advantages are also within the scope of theinvention.

Exemplary flowcharts are provided herein for illustrative purposes andare non-limiting examples of methods. One of ordinary skill in the artwill recognize that exemplary methods may include more or fewer stepsthan those illustrated in the exemplary flowcharts, and that the stepsin the exemplary flowcharts may be performed in a different order thanthe order shown in the illustrative flowcharts.

1. A microparticle for use in a biochemical or chemical assay, themicroparticle comprising: a body comprising a hydrogel, the bodyincluding a probe region and an encoded region; a first plurality ofupconversion nanocrystals disposed in a first portion of the encodedregion, the first plurality of upconversion nanocrystals having a firstspectral signature; and a second plurality of upconversion nanocrystalsdisposed in a second portion of the encoded region spatially separatedfrom the first portion of the encoded region, the second plurality ofupconversion nanocrystals having a second spectral signature differentthan the first spectral signature.
 2. The microparticle of claim 1,wherein the second spectral signature is different than the firstspectral signature.
 3. The microparticle of claim 1, wherein the firstplurality of upconversion nanocrystals includes a first material dopedwith one or more rare earth elements and the second plurality ofupconversion nanocrystals includes a second material doped with one ormore rare earth elements.
 4. The microparticle of claim 1, wherein thebody is asymmetric in shape.
 5. The microparticle of claim 1, whereinthe upconversion nanocrystals are covalently bound to the hydrogelmaterial.
 6. The microparticle of claim 1, wherein the upconversionnanocrystals are bound to the hydrogel material at the time of particlesynthesis through an acrylate group.
 7. The microparticle of claim 1,wherein for each portion of the encoded region, the plurality ofupconversion nanocrystals are dispersed without aggregation.
 8. Themicroparticle of claim 1, further comprising a third plurality ofupconversion nanocrystals disposed in a third portion of the encodedregion spatially separated from the first portion of the encoded regionand spatially separated from the second portion of the encoded region,the third plurality of upconversion nanocrystals having a third spectralsignature different than the first spectral signature.
 9. Themicroparticle of claim 8, wherein the encoded region includes at leastfive different portions.
 10. The microparticle of claim 1, wherein eachspectral signature includes luminescence in multiple distinct bandswithin a range of 400-800 nm.
 11. The microparticle of claim 1, whereinthe probe region comprises polyethylene glycol diacrylate (PEG-DA). 12.The microparticle of claim 1, wherein the encoded region comprisesdi-acrylated monomer.
 13. The microparticle of claim 1, wherein theprobe region comprises a first polymer material and the encoding regioncomprises a second polymer material different than the first polymermaterial.
 14. The microparticle of claim 1, wherein the probe regionincludes one or more molecular recognition elements.
 15. Themicroparticle of claim 1, wherein the upconversion nanocrystals areparamagnetic or ferromagnetic.
 16. A method of making a hydrogelmicroparticle for use in a biochemical or chemical assay, the methodcomprising: providing a first encoded region source material including ahydrogel and a first plurality of upconversion nanocrystals having afirst spectral signature; providing a second encoded region sourcematerial including a hydrogel and a second plurality of upconversionnanocrystals having a second spectral signature; providing a proberegion source material including a hydrogel; cross-linking the firstencoded region source material, the second encoded region sourcematerial and the probe region source material forming a first portion ofan encoded region, a second portion of the encoded region and a proberegion with the probe region crosslinked with one or both of the firstportion and the second portion of the encoded region to form acontiguous microparticle.
 17. The method of claim 16, wherein the secondspectral signature is different than the first spectral signature. 18.The method of claim 16, wherein each of the first plurality ofupconversion nanocrystals and each of the second plurality ofupconversion nanocrystals has a hydrophilic surface.
 19. The method ofclaim 16, wherein the probe region source material includes one or moremolecular recognition elements. 20-25. (canceled)
 26. A method ofperforming a biochemical or chemical assay comprising: exposing a sampleto a plurality of microparticles, each microparticle comprising: a bodycomprising a hydrogel, the body including a probe region with one ormore molecular recognition elements and an encoded region; a firstplurality of upconversion nanocrystals disposed in a first portion ofthe encoded region, the first plurality of upconversion nanocrystalshaving a first spectral signature; and a second plurality ofupconversion nanocrystals disposed in a second portion of the encodedregion spatially separated from the first portion of the encoded region,the second plurality of upconversion nanocrystals having a secondspectral signature different than the first spectral signature; for eachmicroparticle, illuminating the microparticle with an excitation lightsource; for each microparticle, detecting light emitted from theilluminated microparticle, the detected light including upconvertedluminescent light from the first plurality of upconversion nanocrystalsand the second plurality of upconversion nanocrystals and lightassociated with the one or more molecular recognition elements; andidentifying each microparticle based on the detected light. 27-30.(canceled)